This invention relates to the isolation and cloning of genetic information coding for Clostridium histolyticum collagenase and the expression of the genetic information in a suitable host. In particular, this invention is directed to the isolation and cloning of genetic information coding for a single form of Clostridium histolyticum collagenase useful for the reproducible isolation of pancreatic islets from pancreatic tissue.
Patients with Type I diabetes are insulin-dependent because they are unable to produce insulin from the endocrine portion of the pancreas. Whole-pancreas transplantation has been used in an effort to improve the quality of life of diabetic patients and to prevent the long-term complications of the disease such as blindness, peripheral neuropathy, and coronary artery disease.
For insulin-dependent patients, whole-pancreas transplantation has the disadvantage that transplantation of only the endocrine component of the pancreas, the pancreatic islets, is required to correct the insulin deficiency. The remaining exocrine portion of the gland complicates and frequently hampers successful transplantation. Management of the exocrine secretions of the pancreas poses a major technical problem in the treatment of Type I diabetes by whole organ transplantation.
It would, therefore, be desirable to treat diabetes by transplantation of the pancreatic islets instead of the whole pancreas. For that reason, much effort has gone into isolating the islets from the human pancreas for clinical islet transplantation. These efforts have been reviewed in D. W. R. Gray & P. J. Morris, "Prospects for Pancreatic Islet Transplantation," World J. Surg. 10, 410-421 (1986) and D. W. Scharp, "Isolation and Transplantation of Islet Tissue," World J. Surg. 8, 143-151 (1984).
Among the enzymes most suitable for cell isolation is collagenase. Collagenase is a protease specific for the fibrous protein collagen of connective tissue. Collagenase digestion of pancreases for islet cell isolation has been used with some success with pancreases of small animals, but with considerably less success in the more fibrous human pancreas. The clinical results of islet cell transplantation have been poor, as detailed in J. S. Najarian, D. E. Sutherland, A. J. Matas, M. W. Steffes, R. L. Simmons, & F. C. Goetz, "Human Islet Transplantation: A Preliminary Report," Transplant Proc. 9, 233-235 (1977), and D. E. Sutherland, A. J. Matas, F. C. Goetz, & J. S. Najarian, "Transplantation of Dispersed Pancreatic Islet Tissue in Humans: Autografts and Allografts," Diabetes [Supp.] 29, 31-35 (1980). The islet cells obtained have relatively poor viability and are contaminated with exocrine cells.
One of the technical difficulties with human islet isolation stems from inherent lot-to-lot variation in commercially available preparations of collagenase. The underlying reason for this lot-to-lot variation is not understood. Each of the several types of collagenase commercially available requires extensive screening and testing to ensure its efficiency.
Another problem associated with the use of collagenase in human islet cell isolation is that commercial collagenase preparations from C. histolyticum contain highly active non-collagenase proteases that may contribute more proteolytic activity than does the collagenase itself. This further complicates the problem of obtaining a reproducible collagenase preparation for use in islet cell isolation.
The use of Clostridium collagenase for the isolation of pancreatic islets is still further complicated by the existence of multiple forms of collagenase produced by Clostridium. As many as six separate forms of collagenase can be present: .alpha.-collagenase, .beta.-collagenase, .gamma.-collagenase, .delta.-collagenase, .zeta.-collagenase, and .xi.-collagenase. Furthermore, some of these forms are present in subspecies. All of these multiple forms of collagenase are highly active against collagen and devoid of other proteolytic activities. They have molecular weights ranging from 68,000 daltons to 125,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The problem of obtaining preparations of collagenase for reproducible islet cell isolation is further complicated by the fact that many of these forms of collagenase are subject to rapid autocatalytic proteolysis and are degraded to smaller molecules.
The existence of multiple forms of collagenase, contaminating non-collagenase proteases, and rapid autocatalytic proteolytic degradation all contribute to lot-to-lot variation in the activity of commercial Clostridium collagenase. It is not known what factors, such as the strain of Clostridium used, growth medium, growth conditions, or time of harvest, are responsible for this variability. The existence of this variability complicates efforts to use Clostridium collagenase for the isolation of pancreatic islets from human pancreatic tissue because it creates a lack of reproducibility in the isolation of the islets.
Accordingly, there is a need for a method to isolate human islets reproducibly so that the effects of lot-to-lot variations of collagenase enzyme preparations are minimized. In particular, there is a need for preparations of collagenase that are stable and are not subject to autocatalytic degradation for use in islet cell isolation.